Cholecystokinin (CCK) in the GI tract is the major hormonal regulator of pancreatic acinar cell function and gallbladder contraction. CCK is also present in brain where its role is unknown. The objective of the proposed research is to understand the role of the CCK receptor in mediating the action of this hormone. The major aims of the present proposal are threefold. The first will be to isolate, purify, and characterize the CCK receptor from pancreas and brain to obtain its size, chemical composition, and subunit structure. Plasma membranes of pancreatic acini and brain will be purified, the receptors solubilized with detergent, and the solubilized receptor further purified by gel filtration, ion exchange chromatography and affinity chromatography. The purified receptor will be investigated to elucidate its strokes radius, sedimentation coefficient, molecular weight, subunit composition and carbohydrate content. Alternatively, to probe the receptor, radiolabeled CCK will be covalently crosslinked to the receptors on plasma membranes, the membranes solubilized, and the relative molecular weight and subunit structure of the receptor studied by polyacrylamide gel elecytrophoresis. The second aim will be to characterize how CCK receptors and CCK actions are regulated. In vivo studies will be carried out with rats injected daily with CCK for periods of up to two weeks followed by studies of pancreatic function and CCK receptors. In vitro regulation of receptors and their possible recycling within various pancreatic organelles will be probed using phamacologic agents known to inhibit internalization (bacitracin, mechylamine), receptor and hormone degradation (chloroquine) and receptor recycling (monensin). The third aim will be to use isolated pancreatic acini to bioassay and radioreceptor assay CCK in both tissue extracts and plasma. Stimulated amylase release from isolated acini will be used as a bioassay and inhibition of iodinated CCK binding to acini as a radioreceptor assay. These studies will provide data on the hormonal role of CVCK, and its mechanism of action on the exocrine pancreas, gallbladder and brain.